The invention relates to the Duffy blood group protein (also known as gp-Fy) and its function in malaria. More specifically, the invention relates to peptides having amino acid sequences characteristic of Duffy proteins, compositions containing such peptides, and methods of using such compositions to combat malaria
Malaria is the most prevalent infectious disease of mankind. Its widespread geographic distribution together with the severe pathologic consequences of the infection make malaria a major medical and financial burden for many of the developing nations.
There are several different kinds of malaria, one of which is caused by the parasite Plasmodium vivax, which attacks the red blood cells of susceptible individuals. A genetic trait of special interest with regard to P. vivax is the absence of antigens encoded by the blood group system called Duffy (Livingston 1984). It has been shown that individuals whose red blood cells lack the product of the Duffy genes are not susceptible to the penetration of P. vivax owing to the fact that Duffy molecules serve as the receptor for the parasite. (Miller et al. 1976).
Malarial parasites are transmitted from host to host by the feeding females of several species of the genus Anopheles. It is in the mosquito that the sexual phase of the life cycle of P. vivax takes place, leading to the production of sporozoites. After their introduction into a "new" host, these sporozoites reside in the parenchymal cells of the liver and multiply asexually causing the eventual rupture of hepatic cells and the release of asexual forms (merozoites) into the blood stream. The circulating merozoites actively penetrate into red blood cells in a nearly synchronous fashion and, because the rate of growth and cell division of P. vivax is essentially identical, the infected erythrocytes simultaneously reach the stage of parasite load at which they burst. This produces the typical cycles of fever every 48 hours, a condition known as "tertian" malaria. Immunity to P. vivax is commonly only partial in nature, which allows the occurrence of superinfections that evolve independently, causing an overlap in the cycles of parasite release leading to fever cycles of apparently shorter periods.
P. vivax infection may persist without treatment for as long as five years. P. vivax parasitemias are relatively low-grade, primarily because the parasites favor the few young red blood cells or reticulocytes that exist in peripheral blood.
Invasion of erythrocytes by Plasmodium merozoites is a multi-step process that requires a series of specific molecular interactions between the invading merozoite and the target erythrocyte. P. vivax and the related simian malarial parasite, P. knowlesi, both require interaction with the erythrocyte chemokine receptor, also known as the Duffy blood group antigen, to invade human erythrocytes (Miller et al. 1975; Miller et al. 1976; Horuk et al. 1993). The Duffy blood group system consists of two principal antigens Fy.sup.a and Fy.sup.b produced by co-dominant alleles, FY*A and FY*B. Antisera anti-Fy.sup.a and anti-Fy.sup.b have been used to define four phenotypes, Fy(a+b-), Fy(a-b+), Fy(a+b+) and Fy(a-b-) (Marsh 1975). Neither of these antisera agglutinates Duffy Fy(a-b-) cells, the predominant phenotype in Africans and African-Americans. Antisera defining the other Duffy phenotypes, Fy3, Fy4 and Fy5, are very rare. A murine monoclonal antibody, anti-Fy6, defines a Duffy antigenic determinant present in all red blood cells except Fy(a-b-) cells (Nichols 1987). Accordingly, erythrocytes reacting with the Duffy antisera or anti-Fy6 are designated "Duffy-positive," while unreactive erythrocytes are designated "Duffy-negative." Duffy-negative human erythrocytes, which lack the Duffy blood group antigen, are completely resistant to invasion by these parasites.
Unlike P. vivax, however, P. knowlesi can also invade the erythrocytes of rhesus monkeys, which are commonly used as hosts for P. knowlesi in the laboratory. Although P. knowlesi is absolutely dependent on the Duffy blood group antigen for invasion of human erythrocytes, P. knowlesi can use both Duffy antigen-dependent and alternate, independent pathways to invade rhesus erythrocytes (Haynes et al. 1988). The erythrocyte receptors involved in these Duffy antigen-independent invasion pathways of P. knowlesi are not known.
In contrast to P. vivax, P. falciparum can invade both Duffy-positive and -negative human erythrocytes with similar efficiencies. This indicates that P. falciparum does not require the Duffy antigen for invasion. Most P. falciparum strains studied thus far in the laboratory use sialic acid residues on glycophorins as receptors for optimal invasion of human erythrocytes. However, some P. falciparum strains can also use alternate receptors to invade human erythrocytes, although at reduced levels (Mitchell et al. 1986; Hadley et al. 1986; Perkins et al. 1988; Dolan et al. 1994.
The parasite ligands that bind to the several erythrocyte receptors described above belong to a family of erythrocyte binding proteins. This family includes the Duffy-binding proteins of P. vivax and P. knowlesi (designated ".alpha."); the P. knowlesi proteins, designated ".beta." and ".gamma.," which bind as yet unidentified receptors on rhesus erythrocytes; and the P. falciparum protein, designated "EBA-175," which binds sialic acid residues of glycophorin A on human erythrocytes (Adams et al. 1992). The Duffy receptors of P. vivax and P. knowlesi are described in detail in U.S. Pat. Nos. 5,198,347 and 5,541,292 to Miller et al.
Each protein of this family contains two cysteine-rich domains which contain cysteines as well as a number of aromatic amino acid residues that are conserved in position. The functional binding domain of each erythrocyte binding ligand lies in Region II, the 5' cysteine-rich domain of each protein (Chitnis et al. 1994; Sim et al. 1994). These cysteine-rich domains are referred to as Duffy-binding-like (DBL) domains since the first of these domains to be shown to possess adhesive properties was derived from the P. vivax protein that binds the Duffy antigen. DBL domains are also found in the var genes, which encode the variant surface antigens of P. falciparum. They define a large family of Plasmodium proteins, the DBL superfamily, which includes both the erythrocyte binding proteins and the variant surface antigens of Plasmodium (Su et al. 1995; Smith et al. 1995; Baruch et al. 1995).
P. vivax exhibits considerable antigenic diversity and variation, as do other malarial Plasmodium species (Hommel 1985), although it has been recently shown that antigenic components of P. vivax sporozoites exist that are common to parasites from different isolates (Zavala et al. 1985). The merozoites of different strains of P. vivax share the same receptor for penetration into red blood cells, i.e., the Duffy molecule (Miller et al. 1976). Recognizing this feature carries implications for the development of vaccines. In addition, regardless of the parasite's capacity to vary other antigenic molecules, its recognition molecule, i.e., the molecule that binds to the Duffy molecule, must remain constant since it is the complementarily between this molecule and the invariant receptor that allows the penetration of merozoites into erythrocytes and, thus, the continuity of the infection. Changes in the ligand specificity of this molecule would result in the loss of the parasite's capacity to infect, since P. vivax merozoites appear to be unable to utilize other human red blood cell receptors for their penetration in vivo, as shown by the resistance to infection of Duffy-negative erythrocytes.
In view of the above considerations, it is clear that existing knowledge concerning the malarial binding site on erythrocytes has been insufficient to permit molecular approaches to the treatment and prophylaxis of malaria. Lacking an understanding of the molecular basis for the interaction between the merozoite and the Duffy antigen has been a serious impediment preventing the development of compositions and methods by which that interaction, and the concomitant infection, can be effectively inhibited or eliminated.
Accordingly, it is one of the purposes of this invention to overcome the above limitations in the prevention and treatment of malaria, by providing a composition and method capable of inhibiting or preventing the specific parasite-erythrocyte interaction and subsequent infection. Other purposes will present themselves to the skilled artisan to render the invention useful in particular contexts.